CN102746390A - Mycobacterium tuberculosis early culture filtrate protein, its preparation method and application - Google Patents
Mycobacterium tuberculosis early culture filtrate protein, its preparation method and application Download PDFInfo
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Abstract
The invention provides a mycobacterium tuberculosis early culture filtrate protein, its preparation method and an application of the protein in reagents and kits for diagnosing tuberculosis and latent infection people of mycobacterium trberculosis. The preparation method of the protein comprises the following steps: inoculating H37Rv strain to an inclined plane medium, inoculating the cultured strain to a liquid Sauton medium, conducting shaking culturing for 5 days at 37 DEG C, and then separating.
Description
Technical field
The present invention relates to the early stage culturing filtrate albumen of mycobacterium tuberculosis, Preparation Method And The Use, be specifically related to mycobacterium tuberculosis H
37The proteic process for extracting of the early stage culturing filtrate of Rv, and prepared early stage culturing filtrate albumen is in diagnosis of tuberculosis or tubercule bacillus latent infection crowd's purposes.
Background technology
White plaque is to infect the many organ infections property disease that causes by mycobacterium tuberculosis complex, is mainly pulmonary tuberculosis.Whole world tuberculosis infected students is up to 2,000,000,000 people, and annual new cases are about 8,000,000～1,000 ten thousand, and wherein the annual New Development patient of China is up to 1,500,000.The EPDML latent infection that key character is a mycobacterium tuberculosis of tubercule bacillus.The mycobacterium tuberculosis latent infection is a kind of sub-clinical state, no actinoscopy sign, and bacterium is dormant state, has 10% mycobacterium tuberculosis latent infection crowd can develop into tuberculosis patient in life approximately.
Mycobacterium tuberculosis latent infection crowd; Be also referred to as the white plaque high risk population, or be called tubercule bacillus latent infection crowd, usually the test of PPD (Tuberculins,PPD) skin test was strong positive after 72 hours; Diameter >=15mm and/or be called the white plaque high risk population with blister, bad the dead promptly hardens; Be also referred to as mycobacterium tuberculosis high risk population or tubercule bacillus latent infection crowd, for for simplicity, the application is referred to as tubercule bacillus latent infection crowd with it.
China belongs to one of the serious popular of white plaque country in the world; Whole nation tuberculosis epidemiology investigation shows; The population of China about 1/3rd has infected tubercule bacillus, and infected number surpasses 400,000,000, and wherein some people will develop into active tuberculosis.Therefore something must be done to control effectively, and prevention latent infection crowd develops into tuberculosis patient, and how effective, comprehensive examination goes out the latent infection crowd, then is the primary problem that solves.Tubercule bacillus latent infection crowd does not still have the diagnosis gold standard at present; The test of PPD skin test is diagnosing tubercle bacillus latent infection crowd's a domestic method; But this method has certain defective; Because test used PPD antigen is that mycobacterium tuberculosis, non-virulent mycobacterium and BCG-CWS (being BCG) are common, has cross reaction, and specificity is poor.China is the country of general bcg vaccination; And because BCG vaccination person and tubercule bacillus natural infection person upward are difficult to differentiation to the performance that is reflected at that PPD tests; Even adopt above-mentioned PPD skin test test strong positive as judgement criteria; Still can some BCG vaccination person and the infected of non-virulent mycobacterium included among the tubercule bacillus latent infection crowd, therefore cause the practical function of PPD test in tubercule bacillus latent infection crowd diagnosis very limited.The epidemic situation was severe for China's white plaque; And the latent infection crowd is big, scope extensively is an important factor that causes white plaque to be broken out at present; If can diagnose out tubercule bacillus latent infection crowd comprehensively and effectively and can and intervene, just can effectively prevent generation lungy treating in early days of infecting.Therefore seek suitable tubercle bacillus specific antigen and new diagnostic techniques, the country of the so general bcg vaccination of object China will be very necessary, also particularly important.
IFN-discharges analytical technology (interferon gamma release assays; IGRA), be a kind of in-vitro diagnosis method that is used for mycobacterium tuberculosis infection, its principle is after the organism infection tubercule bacillus; The effector T cell that is stored in the blood can be when contacting tubercle bacillus specific antigen once more; Produce and the secrete cytokines IFN-,, can diagnose white plaque or tuberculosis infection through detection by quantitative to the specific effector cell of IFN-or generation IFN-.Enzyme-linked immunosorbent assay (ELISA) is used in the detection of IFN-usually, and existing commercial kit QuantiFERON-TB (being called for short TB Gold) is adopting said method quantitative analysis specificity IFN-
YLevel.Detection by quantitative to the specific effector cell that produces IFN-is then used enzyme linked immunological spotting method (ELISPOT), and existing commercial test kit TB-SPOT.TB is an adopting said method, at the T effector cell of unicellular horizontal detection by quantitative secretion IFN-.
Above-mentioned two kinds of diagnostic products have all been selected for use and only have been present in the mycobacterium tuberculosis genome, but the RD1 district proteins encoded that in BCG-CWS and non-virulent mycobacterium, generally lacks is as specific T cell antigen.What antigen all adopted is that traditional synthetic peptide loads the method that stimulates; Even as the stimulus that detects sample, used specific antigens mainly is ESAT-6 and the CFP-10 in the early stage secretory protein of mycobacterium tuberculosis with the overlapped synthetic peptide mixture that derives from specific antigens.But this method also has its limitation; Be that the mixed polypeptide pond can not comprise the epitope peptide that mycobacterium tuberculosis is whole; In the detection of total man's monoid, still there is deficient phenomena, can causes to detect to omit to produce false negative, special in the such situation that the epidemic situation was severe, the latent infection scope is big of China; Can as far as possible comprehensively detect latent infection person has its important practical sense, and there is limitation in the mentioned reagent box on the one hand at this.
Secreted protein antigen in the early stage culturing filtrate of mycobacterium tuberculosis is memory immunity CD4
+The target molecule of T cell.Usually the filtrating albumen that cultivation is no more than 7 days is called early stage culturing filtrate albumen, and said early stage culturing filtrate albumen comprises multiple antigen protein, for example possibly have the albumen such as ESAT6, MPT64 and Ag85b that identified.In the mycobacterium tuberculosis culturing process; Along with the increase of cultivating fate; Protein concentration in the filtrating albumen and/or composition are also changing; The discovery that the present inventor is surprised, the filtrating albumen of cultivating 5 days has excellent more effect than cultivation 7 days and above filtrating albumen aspect differentiation BCG vaccination and the mycobacterium tuberculosis infection, and this effect has no report to disclose or enlighten.And the contriver proves 5 days filtrating albumen of cultivation and PPD skin test strong positive through experiment, and consistence is high as a result; Show that it can detect tubercule bacillus latent infection crowd widely than the commercially available prod; Therefore to cultivate 5 days early stage culturing filtrate albumen as stimulus; Preferred combination ELISPOT diagnostic techniques; Be more suitable in tubercule bacillus latent infection crowd's examination; It has higher detection sensitivity simultaneously and specificity can guarantee that more infection population is detected, and effectively reduces BCG vaccination person's interference, has important practical significance and wide application prospect in the country of the high like this infection of China, high morbidity and general bcg vaccination.
Summary of the invention
The invention provides a kind of have sensitivity preferably and specificity, with PPD skin test strong positive high, the early stage culturing filtrate albumen of mycobacterium tuberculosis that is suitable for white plaque or tubercule bacillus latent infection crowd examination of consistence as a result.This early stage culturing filtrate albumen is that mycobacterium tuberculosis is cultivated the filtrating albumen that obtained in 5 days, the preferred H of said mycobacterium tuberculosis
37Rv mycobacterium (commercially available); The preferred shaking culture of said culture condition, the logical substratum of substratum preferred liquid Soviet Union of said cultivation, said cultivation is carried out after being preferable over activation culture; Said activation culture is preferable on the slant medium carries out; The preferred Luo Shi egg medium of said slant medium, said filtrating albumen preferably obtain the centrifugal concentrated substratum metathetical step that preferably includes of said ultrafiltration by the bacterium liquid of cultivating after 5 days through centrifugal, filtration, the centrifugal concentrated back of ultrafiltration.
The mycobacterium tuberculosis that the present invention the mentions albumen of filtrating in early days refers in particular to and cultivates the filtrating albumen that obtained in 5 days.
Further, the present invention provides mycobacterium tuberculosis early stage culturing filtrate albumen, and its preparation method comprises the steps:
1) strain activation and culture: get H
37The Rv mycobacterium is inoculated in slant medium and cultivates, and collects cultured thalline;
2), be inoculated in the logical substratum of liquid Soviet Union 37 ℃ of shaking culture 5 days with the logical resuspended thalline of substratum of liquid Soviet Union;
3) get the bacterium liquid of cultivating 5 days and carry out spinning, collect culture supernatant, the culture supernatant filtration sterilization;
4) it is centrifugal concentrated the sterile culture supernatant to be carried out ultrafiltration, obtains early stage culturing filtrate albumen of the present invention.
Above-mentioned early stage culturing filtrate albumen of the present invention, wherein the 1st) step strain activation and culture temperature is preferred 35-37 ℃, and more preferably 37 ℃, cultivated 21 days, the common experimenter of this area can judge well-grown, free of contamination thalline; The preferred Luo Shi egg of substratum slant medium; The microorganism collection method preferably washes with saline water, centrifugal collection thalline; Before with the logical resuspended thalline of substratum of liquid Soviet Union, preferably to revive and lead to the thalline that the substratum washing is collected with liquid, washing methods is that the logical substratum of liquid make-up Soviet Union arrives centrifugal preceding volume, centrifugal again collection thalline preferably carries out 3 washings; Step 2) inoculum density preferred 1 * 10
6Individual cell/ml～1 * 10
8Individual cell/ml, preferred 1 * 10
7Individual cell/ml; The optimum condition of shaking culture is 100-200rpm; Preferably be inoculated in the triangular flask that the logical substratum of 200ml liquid Soviet Union is housed of 500ml; The optimum condition of step 3) spinning is 0-8 ℃, and 4000-10000rpm, centrifugation time are 10-60min, more preferably 4 ℃, and 8000rpm, centrifugation time 30min; Filtration sterilization can be used the method for conventional protein filtration sterilization, preferably uses the filter membrane of 0.22 μ m; The centrifugal spissated optimum condition of step 4) ultrafiltration is 0-8 ℃, 2000-6000rpm, more preferably 4 ℃, 4000rpm; Step 4) is preferably carried out the sterile culture supernatant that ultrafiltration is centrifugal to be concentrated into 5-15 doubly; Be preferably 10 times, add pH6.5-7.5 then, preferred pH is that 7.0 phosphate buffered saline buffer is to original volume; It is centrifugal concentrated to carry out ultrafiltration under the same terms; Repeat to add phosphate buffered saline buffer, the centrifugal enrichment step of ultrafiltration 1-4 time again, preferred 3 times, obtain early stage culturing filtrate albumen.
The present invention also provides the early stage culturing filtrate of mycobacterium tuberculosis proteic preparation method, may further comprise the steps:
1) strain activation and culture: get H
37The Rv mycobacterium is inoculated in slant medium and cultivates, and collects cultured thalline;
2), be inoculated in the logical substratum of liquid Soviet Union 37 ℃ of shaking culture 5 days with the logical resuspended thalline of substratum of liquid Soviet Union;
3) get the bacterium liquid of cultivating 5 days and carry out spinning, collect culture supernatant, the culture supernatant filtration sterilization;
4) it is centrifugal concentrated the sterile culture supernatant to be carried out ultrafiltration, obtains early stage culturing filtrate albumen of the present invention.
Above-mentioned preparation method provided by the invention, wherein the 1st) step strain activation and culture temperature is preferred 35-37 ℃, and preferred 37 ℃, cultivated 21 days, the common experimenter of this area can judge well-grown, free of contamination thalline; The preferred Luo Shi egg of substratum slant medium; The microorganism collection method preferably washes with saline water, centrifugal collection thalline; Before with the logical resuspended thalline of substratum of liquid Soviet Union, preferably to revive and lead to the thalline that the substratum washing is collected with liquid, washing methods is that the logical substratum of liquid make-up Soviet Union arrives centrifugal preceding volume, centrifugal again collection thalline preferably carries out 3 washings; Step 2) inoculum density preferred 1 * 10
6Individual cell/ml～1 * 10
8Individual cell/ml, preferred 1 * 10
7Individual cell/ml; The optimum condition of shaking culture is 100-200rpm; Preferably be inoculated in the triangular flask that the logical substratum of 200ml liquid Soviet Union is housed of 500ml; The optimum condition of step 3) spinning is 0-8 ℃, and 4000-10000rpm, centrifugation time are 10-60min, more preferably 4 ℃, and 8000rpm, centrifugation time 30min; Filtration sterilization can be used the method for conventional protein filtration sterilization, preferably uses the filter membrane of 0.22 μ m; The centrifugal spissated optimum condition of step 4) ultrafiltration is 0-8 ℃, 2000-6000rpm, more preferably 4 ℃; 4000rpm, step 4) is preferably carried out the sterile culture supernatant that ultrafiltration is centrifugal to be concentrated into 5-15 doubly, is preferably 10 times; Add pH6.5-7.5 then, preferred pH be 7.0 phosphate buffered saline buffer to original volume, carry out under the same terms that ultrafiltration is centrifugal to be concentrated; Repeat to add phosphate buffered saline buffer, the centrifugal enrichment step of ultrafiltration 1-4 time again, preferred 3 times, obtain early stage culturing filtrate albumen.
Among the proteic preparation method of early stage culturing filtrate of the present invention; Preferred substratum displacement step; Promptly the 4th) step in the step that repeats to add phosphate buffered saline buffer, ultrafiltration and concentration; Like this can enough phosphate buffered saline buffers replace the most of medium component in the culture supernatant, make the early stage culturing filtrate protein ingredient of acquisition simple, purity is higher.
The proteic preparation method of early stage culturing filtrate of the present invention washs thalline before preferably including the inoculation liquid nutrient medium; Along with increasing of wash number; The protein content that contains in the bacterial classification inoculation liquid reduces gradually; Can remove thalline itself remaining secretory protein and thalline membranin, help to improve the target proteic purity of filtrating.
The present invention also provide the early stage culturing filtrate albumen of 5 days mycobacterium tuberculosis of above-mentioned cultivation in preparation white plaque or tubercule bacillus latent infection diagnostic reagent or the purposes in the test kit and comprise the proteic diagnostic kit of said filtrating.The IFN-that it is stimulus that said diagnostic reagent or test kit are preferably with said early stage culturing filtrate albumen
Y-ELISPOT diagnostic reagent and test kit, the early stage preferred 5 μ g/ml of the proteic dosage of culturing filtrate in said diagnostic reagent or the test kit.
Tubercule bacillus latent infection crowd does not still have the diagnosis gold standard at present; And the present invention is through the surprised discovery of research; The examination that the early stage culturing filtrate albumen of cultivating 5 days of mycobacterium tuberculosis is more suitable for tubercule bacillus latent infection crowd than early stage culturing filtrate albumen and the existing commercial reagent box cultivated 7 days; Cultivate 5 days early stage filtrating albumen and can better distinguish BCG inoculation and mycobacterium tuberculosis infection than the early stage filtrating albumen of cultivating 7 days; Thereby overcome the defective of PPD test diagnosis poor specificity, cultivate early stage filtrating albumen-INF-of 5 days simultaneously
Y-ELISPOT method and PPD skin test strong positive result have very high consistence (93.7%); And existing T-SPOT.TB test kit consistence is merely 50%; Show that the early stage filtrating albumen examination widely of cultivating 5 days goes out tubercule bacillus latent infection person, the early stage culturing filtrate albumen of therefore cultivating 5 days is more suitable for tubercule bacillus latent infection crowd's examination.
Embodiment:
Embodiment 1: the early stage proteic preparation of culturing filtrate
Present embodiment has specified H
37Cultivation of Rv mycobacterium and the early stage proteic preparation method of culturing filtrate.
1.1H
37Cultivation of Rv mycobacterium and inoculation
With H
37Rv mycobacterium (ATCC 93009) is inoculated on the Luo Shi egg slant medium, cultivates 21 days for 37 ℃.Get inclined-plane well-grown and free of contamination thalline, wash bacterial classification, be collected in the centrifugal barrel with saline water, 4 ℃, 8000rpm, centrifugal 30min discards culture supernatant after the centrifugal end, collects thalline.Wash thalline 3 times with the logical substratum of liquid Soviet Union; Washing methods is that liquid make-up is revived logical substratum to centrifugal preceding volume, centrifugal again collection thalline, and protein content is 547 μ g/ml in the supernatant of cleaning back for the first time; Protein content is 147 μ g/ml in the supernatant of cleaning back for the second time; Protein content is 64 μ g/ml in the supernatant of cleaning back for the third time, and hence one can see that, and preceding thalline is washed of inoculation can effectively be removed remaining secretory protein and thalline membranin etc.Get the thalline after the resuspended washing of substratum is led in liquid Soviet Union, by 1 * 10
7The inoculum density of individual cell/ml is inoculated in the triangular flask that the logical substratum of 200ml liquid Soviet Union is housed of 500ml, 100～200rpm, and 37 ℃ of concussions are cultivated.
1.2 proteic collection of culturing filtrate and preparation
The mycobacterium tuberculosis bacterium liquid of cultivating 0,3,5,7,10,13,15 day is collected in the centrifugal barrel, 4 ℃, 8000rpm, centrifugal 30min discards thalline after the centrifugal end, collects culture supernatant.Culture supernatant is crossed 0.22 μ m membrane filtration degerming.Culture supernatant after the Sterile Filtration is carried out the centrifugal concentrated and displacement damping fluid of ultrafiltration.
The ultra-filtration centrifuge tube of experiment condition: 5000bp or ultrafiltration Centrifuge Cup, millipore company; Refrigerated centrifuge, eppendorf company.
Experiment reagent: the pH value is 7.0 phosphate buffered saline buffer.
Experimental procedure: it is centrifugal at first to place ultra-filtration centrifuge tube or Centrifuge Cup to carry out the sterile culture supernatant, and centrifugal condition is 4 ℃, 4000rpm, and the control centrifugation time makes culture supernatant concentrate 10 times; Add phosphate buffered saline buffer then to original volume, the same terms carries out centrifugal concentrated 10 times of ultrafiltration; Repeat to add phosphate buffered saline buffer, the centrifugal enrichment step of ultrafiltration 3 times after the centrifugal end again, the medium component in the culture supernatant is removed, be replaced as phosphate buffered saline buffer; Concentrate the albumen that obtains at last and be culturing filtrate albumen.Wherein cultivate the filtrating albumen that obtained in 5 days and be called early stage culturing filtrate albumen.Can adjust protein concentration according to using dosage during use.
Embodiment 2: the culturing filtrate protein concentration of different incubation time results and diagnosis effect thereof are relatively
2.1 determining the protein quantity
With the Lowry method carry out protein content mensuration (referring to document Loery OH, Rosebrough NJ, FarrAL, et al.Protein measurement with the folin phenol reagent.J BiolChem.1951,193 (1): 265-275).
Protein concentration is following in the culturing filtrate of the different incubation time results that embodiment 1 obtains:
0 day culturing filtrate protein concentration: 18 μ g/ml
3 days culturing filtrate protein concentrations: 33 μ g/ml
5 days culturing filtrate protein concentrations: 51 μ g/ml
7 days culturing filtrate protein concentrations: 61 μ g/ml
10 days culturing filtrate protein concentrations: 72 μ g/ml
13 days culturing filtrate protein concentrations: 188 μ g/ml
15 days culturing filtrate protein concentrations: 222 μ g/ml
2.2 cultivate 5 days early stage filtrating protein stability analysis
Can know that by embodiment 2.1 results along with the prolongation of incubation time, mycobacterium tuberculosis secretory is also different to protein concentration and/or composition in the culturing filtrate supernatant behind the different incubation times.So far; Though having research from said filtrating albumen, to isolate some single albumen analyzes; But be not difficult to clearly determine all albumen and the exact concentration in the mycobacterium tuberculosis culturing filtrate albumen so far yet; Therefore this area all is to adopt the cultivation fate to characterize for the sign of mycobacterium tuberculosis culturing filtrate protein composition, can clearerly confirm said protein composition like this.And one skilled in the art will appreciate that bacterial classification inoculation with active state behind liquid nutrient medium, the growth curve of different batches is close; Thalline also is relative constant to the secretion time of specific protein, and the contriver adopts the method for embodiment 1 to repeat repeatedly to test, and the result cultivates 5 days filtrating protein concentration all about 50 μ g/ml; Promptly confirm under certain thalline inoculum size and culture condition; Protein concentration is relatively stable in the filtrating of fixing cultivation fate, and its difference belongs to the acceptable limit of error in this area, therefore; Characterizing filtrating albumen with incubation time can the said protein composition of clearer sign, and the realization of its function is not exerted an influence.
2.3 the proteic diagnosis effect of culturing filtrate of different incubation time results relatively
Culturing filtrate albumen is diluted, and the proteic performance of with the dosage of 3 μ g/ml different incubation times being gathered in the crops respectively of culturing filtrate compares.Experimental technique: choose tuberculosis patient (be called for short patient) and the no tuberculosis contact history of clinical definite and inoculated healthy volunteer's (being called for short healthy inoculator) of BCG; The in-vitro separation PMNC; Mononuclearcell is through counting and after mixing up cell concn, and culturing filtrate albumen that obtains with the different incubation times of negative control thing (substratum), 3 μ g/ml respectively and positive control (PHA) are at 5%CO
2, hatched jointly 16-20 hour under 37 ℃ of conditions.Discard nutrient solution after hatching end, repeat to wash plate 4 times with 200 μ LPBS damping fluids.Every hole adds 50 μ L traget antibody working fluids, puts 2～8 ℃ and hatches 60 minutes.Discard liquid in the reacting hole after hatching end, repeat to wash plate 4 times with 200 μ L PBS damping fluids.Every hole adds chromogenic substrate solution 50 μ L, reacts at room temperature after 5～10 minutes to form spot.With each hole termination reaction of experimental water washing.
Through forming spot after above-mentioned antigen-antibody binding reaction and the enzymatic reaction, (positive judgement criteria is counting spot number: experimental port spot number >=6) to diagnose.Comparative result is seen table 1.
Table 1: the culturing filtrate albumen-INF-of different incubation time results
YThe diagnostic result of-ELISPOT
(explain: "+" expression is positive, and "-" expression is negative)
Result by last table; The discovery that the contriver is surprised; Cultivating the early stage culturing filtrate albumen that obtained in 5 days can better distinguish with the healthy volunteer who inoculated BCG tuberculosis patient; Cultivated 3 days or shorter time and cultivated 7 days or the longer time culturing filtrate albumen then can not distinguish or specificity low; Such difference and effect have no report to disclose or enlighten in research in the past, though prove and only be separated by two days, the early stage culturing filtrate albumen of cultivating 5 days has the unforeseeable effect of filtrating albumen of cultivating 3 days and 7 days.
Embodiment 3:5 days early stage culturing filtrate albumen is as IFN-
Y-ELISPOT experiment is with the dosage markization of stimulus
3.1 early stage culturing filtrate albumen-IFN-of 5 days of different concns
YThe Study of Sensitivity of-ELISPOT diagnosis
Tuberculosis patient 24 examples of clinical definite are chosen in experiment; 5 days the early stage culturing filtrate albumen of cultivation that obtains with the embodiment of different concns 1 is that stimulus carries out external ELISPOT diagnosis (concrete grammar is with embodiment 2; Dosage adapts to adjustment), low to study (0.2 μ g/ml), in the early stage culturing filtrate albumen of (1 μ g/ml), high different concns such as (5 μ g/ml) at IFN-
YSensitivity differences in the-ELISPOT diagnostic method.Experimental result is seen table 2.
Table 2: different concns is cultivated early stage culturing filtrate albumen-IFN-of 5 days
YThe interpretation of result of-ELISPOT diagnosis in tuberculosis patient
3.2 early stage culturing filtrate albumen-IFN-of 5 days of different concns
YThe The specificity of-ELISPOT diagnosis
20 of the healthy volunteers of the negative no white plaque contact history of PPD skin test are chosen in experiment, and 5 days the early stage culturing filtrate albumen of cultivation that obtains with the embodiment 1 of different concns carries out external IFN-as stimulus
Y-ELISPOT diagnoses (concrete grammar is with embodiment 2, and dosage adapts to adjustment), low to study (0.2 μ g/ml), in (1 μ g/ml), the early stage culturing filtrate albumen-IFN-of high (5 μ g/ml) different concns
YThe specificity of-ELISPOT diagnosis.Experimental result is seen table 3.
Table 3: different concns is cultivated early stage culturing filtrate albumen-IFN-of 5 days
YThe interpretation of result of-ELISPOT diagnosis in the healthy volunteer
3.3 early stage culturing filtrate albumen-IFN-
YThe selection of optimal stimulation source dosage in the-ELISPOT diagnostic method
The different target crowd is chosen in above-mentioned experiment, that is, the healthy volunteer of the no tuberculosis contact history that the tuberculosis patient of clinical definite and PPD test are negative carries out external early stage culturing filtrate albumen-INF-
Y-ELISPOT diagnosis has been compared as stimulus, and the early stage proteic different concns of culturing filtrate carries out INF-
YThe susceptibility of-ELISPOT in-vitro diagnosis and specificity difference.According to experimental result, optimize the proteic irritating concentration of early stage culturing filtrate, confirm early stage culturing filtrate the righttest proteic working concentration, set up standardized early stage culturing filtrate albumen-INF-
Y-ELISPOT diagnostic method, the experimental result of acquisition is seen table 4.
Table 4: the early stage culturing filtrate albumen-INF-of different concns
YThe susceptibility and the specificity of-ELISPOT diagnosis are summed up
Early stage culturing filtrate albumen-INF-according to different concns
Y-ELISPOT diagnostic result is chosen all higher diagnostic bank of susceptibility and specificity, confirms that early stage culturing filtrate the righttest proteic working concentration is 5 μ g/ml.
Embodiment 4: cultivate early stage culturing filtrate albumen-INF-of 5 days
Y-ELISPOT method is used for examination tubercule bacillus latent infection crowd's research
Tubercule bacillus latent infection crowd does not still have the diagnosis gold standard, and the crowd who usually PPD skin test result is strong positive (scleroma diameter >=15mm and/or with blister, necrosis) thinks tubercule bacillus latent infection crowd.PPD skin test strong positive crowd is chosen in experiment, i.e. 72 hours scleroma diameter >=15mm and/or blister, bad the dead are arranged behind the PPD skin test, and this part crowd should be the skin test reaction strong positive that mycobacterium tuberculosis infection causes in theory.Dosage with 5 μ g/ml carries out early stage culturing filtrate albumen-INF-of 5 days that embodiment 1 obtains to this part crowd
Y-ELISPOT diagnoses (concrete grammar is with embodiment 2), and compares with T SPOT.TB result simultaneously.Experimental result is seen table 5.
Generally speaking, crowd PPD skin test negative, the weak positive of reaction or the positive behind the inoculation BCG, promptly 72 hours scleroma diameter≤15mm behind the skin test do not have bubble or necrosis.Therefore; PPD skin test strong positive crowd is chosen in this experiment; Be 72 hours scleroma diameter >=15mm and/or blister, bad the dead are arranged behind the PPD skin test; This part crowd should be the skin test reaction strong positive that m tuberculosis infection causes in theory, and also possibly be to be caused by BCG inoculation or non-pathogenic mycobacterium in the middle of the practice, but possibility is less.
Table 5: early stage culturing filtrate albumen-INF-
Y-ELISPOT method and T SPOT.TB are at tubercule bacillus latent infection crowd's diagnostic result and PPD skin test result's consistency analysis
Interpretation of result:
The result of table 5 shows, early stage culturing filtrate albumen-INF-of 5 days of cultivation of the present invention
Y-ELISPOT method and PPD skin test strong positive result have good consistence, and the coincidence rate of T SPOT.TB is very low.Because the PPD skin test can detect tuberculosis patient and mycobacterium tuberculosis infection crowd widely; And the crowd of the PPD skin test strong positive that this experiment is chosen has got rid of most of BCG inoculator; Therefore having good consistence with PPD skin test strong positive result shows that 5 days early stage culturing filtrate albumen examination widely of cultivation of the present invention goes out tubercule bacillus latent infection crowd, remedies the omission that T SPOT.TB exists.
Infect and the high country of falling ill owing to China is that tuberculosis is high, need comprehensive tubercule bacillus latent infection crowd examination means, experiment of the present invention shows the early stage culturing filtrate albumen-INF-that cultivated 5 days
Y-ELISPOT method is more efficient than T SPOT.TB test kit, and at the so general bcg vaccination of China, and the country with very high erythema nodosum and sickness rate has important practical significance and wide application prospect.
Claims (10)
1. the early stage culturing filtrate albumen of mycobacterium tuberculosis is characterized in that this early stage culturing filtrate albumen is the culturing filtrate albumen that mycobacterium tuberculosis was cultivated 5 days.
2. the early stage culturing filtrate albumen of mycobacterium tuberculosis as claimed in claim 1 is characterized in that, it is obtained by the preparation method who may further comprise the steps:
1) strain activation and culture: get H
37The Rv mycobacterium is inoculated in slant medium and cultivates, and collects cultured thalline; Said slant medium is preferably Luo Shi egg slant medium, and culture condition is preferably under 37 ℃ of conditions and cultivated 21 days, and the microorganism collection method is preferably with saline water and washes, centrifugal collection thalline;
2), be inoculated in the logical substratum of liquid Soviet Union 37 ℃ of shaking culture 5 days with the logical resuspended thalline of substratum of liquid Soviet Union; Preferably before with the logical resuspended thalline of substratum of liquid Soviet Union, wash the thalline of collecting with the logical substratum of liquid Soviet Union; Preferred inoculum density is 1 * 10 during inoculation
6Individual cell/ml～1 * 10
8Individual cell/ml, more preferably 1 * 10
7Individual cell/ml; Shaking culture condition optimization 100-200rpm; Preferably be inoculated in the triangular flask that the logical substratum of 200ml liquid Soviet Union is housed of 500ml;
3) get the bacterium liquid of cultivating 5 days and carry out spinning, collect culture supernatant, the culture supernatant filtration sterilization; The condition optimization of spinning is centrifugal 30min under 4 ℃, 8000rpm condition, and the filter membrane of 0.22 μ m is preferably used in filtration sterilization;
4) it is centrifugal concentrated the sterile culture supernatant to be carried out ultrafiltration, obtains early stage culturing filtrate albumen of the present invention; Carry out under 4 ℃, the condition of 4000rpm preferably that said ultrafiltration is centrifugal to be concentrated.
3. the early stage culturing filtrate albumen of mycobacterium tuberculosis as claimed in claim 2 is characterized in that, the centrifugal simmer down to of the ultrafiltration of step 4) with the centrifugal concentrated 5-15 of said sterile culture supernatant ultrafiltration doubly; Preferred concentrated 10 times, add pH6.5-7.5 then, the phosphate buffered saline buffer of preferred pH7.0 is to original volume; It is centrifugal concentrated to carry out ultrafiltration under the same terms; Repeat to add phosphate buffered saline buffer, the centrifugal spissated step of ultrafiltration 1-4 time again, preferably repeat 3 times, obtain early stage culturing filtrate albumen.
4. the proteic preparation method of the early stage culturing filtrate of the described mycobacterium tuberculosis of claim 1 is characterized in that may further comprise the steps:
1) strain activation and culture: get H
37The Rv mycobacterium is inoculated in slant medium and cultivates, and collects cultured thalline; Said slant medium is preferably Luo Shi egg slant medium, and culture condition is preferably under 37 ℃ of conditions and cultivated 21 days, and the microorganism collection method is preferably with saline water and washes, centrifugal collection thalline;
2), be inoculated in the logical substratum of liquid Soviet Union 37 ℃ of shaking culture 5 days with the logical resuspended thalline of substratum of liquid Soviet Union; Preferably before with the logical resuspended thalline of substratum of liquid Soviet Union, wash the thalline of collecting with the logical substratum of liquid Soviet Union; Preferred inoculum density is 1 * 10 during inoculation
6Individual cell/ml～1 * 10
8Individual cell/ml, more preferably 1 * 10
7Individual cell/ml; Shaking culture condition optimization 100-200rpm; Preferably be inoculated in the triangular flask that the logical substratum of 200ml liquid Soviet Union is housed of 500ml;
3) get the bacterium liquid of cultivating 5 days and carry out spinning, collect culture supernatant, the culture supernatant filtration sterilization; The condition optimization of spinning is centrifugal 30min under 4 ℃, 8000rpm condition, and the filter membrane of 0.22 μ m is preferably used in filtration sterilization;
4) it is centrifugal concentrated the sterile culture supernatant to be carried out ultrafiltration, obtains early stage culturing filtrate albumen of the present invention; Carry out under 4 ℃, the condition of 4000rpm preferably that said ultrafiltration is centrifugal to be concentrated.
5. the proteic preparation method of the early stage culturing filtrate of mycobacterium tuberculosis as claimed in claim 4; It is characterized in that the centrifugal simmer down to of the ultrafiltration of step 4) with the centrifugal concentrated 5-15 of said sterile culture supernatant ultrafiltration doubly preferably concentrates 10 times; Add pH6.5-7.5 then; The phosphate buffered saline buffer of preferred pH7.0 is to original volume, and it is centrifugal concentrated to carry out ultrafiltration under the same terms, repeats to add phosphate buffered saline buffer, the centrifugal spissated step of ultrafiltration 1-4 time again; The preferred repetition 3 times obtains early stage culturing filtrate albumen.
6. the early stage culturing filtrate albumen of each described mycobacterium tuberculosis of claim 1-3 is in preparation white plaque or the diagnostic reagent of tubercule bacillus latent infection or the application in the diagnostic kit.
7. the early stage culturing filtrate albumen of mycobacterium tuberculosis for preparing of each preparation method of claim 4-5 is in preparation white plaque or the diagnostic reagent of tubercule bacillus latent infection or the application in the diagnostic kit.
8. according to each described application of claim 6-7; Wherein diagnostic reagent is with each described early stage culturing filtrate albumen of claim 1-3, or the early stage culturing filtrate albumen for preparing with each described preparation method of the claim 4-5 IFN-that is stimulus
Y-ELISPOT diagnostic reagent, the preferred early stage proteic concentration of culturing filtrate is 5 μ g/ml.
9. the diagnostic kit that is used for diagnosis of tuberculosis or tubercule bacillus latent infection; It is characterized in that; The early stage culturing filtrate albumen of mycobacterium tuberculosis for preparing with each preparation method of the early stage culturing filtrate albumen of each described mycobacterium tuberculosis of claim 1-3 or claim 4-5 is stimulus, and the preferred early stage proteic concentration of culturing filtrate is 5 μ g/ml.
10. diagnostic kit according to claim 9 is characterized in that said diagnostic kit comprises IFN-
Y-ELISPOT diagnostic reagent.
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| CN104027802A (en) * | 2014-05-09 | 2014-09-10 | 中国食品药品检定研究院 | Reinforced tuberculosis subunit vaccine |
| CN108918890A (en) * | 2018-07-23 | 2018-11-30 | 北京市结核病胸部肿瘤研究所 | The method for screening the ligandin of the people of selectively targeted mycobacterium tuberculosis |
| CN109182167A (en) * | 2018-08-15 | 2019-01-11 | 北京祥瑞生物制品有限公司 | A kind of high-titer tuberculin skin test diagnostic reagent (PPD) production technology |
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| CN113444671A (en) * | 2021-08-04 | 2021-09-28 | 成都可恩生物科技有限公司 | Subculturing method of bacillus calmette-guerin strain capable of replacing culture medium of sutong potato |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104027802A (en) * | 2014-05-09 | 2014-09-10 | 中国食品药品检定研究院 | Reinforced tuberculosis subunit vaccine |
| CN108918890A (en) * | 2018-07-23 | 2018-11-30 | 北京市结核病胸部肿瘤研究所 | The method for screening the ligandin of the people of selectively targeted mycobacterium tuberculosis |
| CN109182167A (en) * | 2018-08-15 | 2019-01-11 | 北京祥瑞生物制品有限公司 | A kind of high-titer tuberculin skin test diagnostic reagent (PPD) production technology |
| CN112813121A (en) * | 2021-02-02 | 2021-05-18 | 成都可恩生物科技有限公司 | Method for preparing tuberculin pure protein derivative by using mycobacterium tuberculosis low virulent strain H37Ra |
| CN113444671A (en) * | 2021-08-04 | 2021-09-28 | 成都可恩生物科技有限公司 | Subculturing method of bacillus calmette-guerin strain capable of replacing culture medium of sutong potato |
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